Neural stem cells (NSC) are primordial uncommitted cells postulated to give rise to the array of specialized cells in the CNS. Following implantation in vivo, these cells intermingle with endogenous progenitors and respond to local cues, appropriately differentiating into diverse neuronal and glial types. They have been shown to develop into cells of most neuronal and glial lineage and have the potential for treating a wide variety of neurodegenerative conditions. Transplantation of NSC could also be of benefit in degenerative conditions of the enteric nervous system such as achalasia. Preliminary experiments in our laboratory have demonstrated that NSC isolated from fetal rat forebrain (CNS-NSC) can, not only express nNOS and produce NO in vitro, but also be successfully transplanted into the pylorus wall of mice, where they differentiate into neurons, continue to express nNOS and survive up to 8 weeks post-grafting. We have also shown that rat CNS-NSC are able to form functional neuromuscular connections in vitro and may be able to modulate gastric emptying in vivo. These studies therefore support the potential use of CNS-NSC transplantation in the gastrointestinal tract as a promising cellular replacement strategy for enteric neurons. Having established the feasibility of neuronal transplantation into the gut, we now wish to study the optimal conditions required for successful engraftment and functional benefit of CNS-NSC. In this proposal, we will use CNS-NSC derived from embryonic transgenic LacZ mice. We have shown that these cells are multipotent, express nNOS and produce NO and can be easily tracked after transplantation in vivo. In addition they express the receptor RET and proliferate in response to the enteric neurotrophin GDNF. Our hypothesis is that CNS-NSC can survive transplantation in the gut and undergo differentiation into functional enteric neurons. To test this hypothesis we will pursue the following specific aims: SPECIFIC AIM 1: To determine the post-implantation fate (survival and differentiation) of transplanted CNS-NSC, and to assess the benefit, if any, of immunosuppressive therapy in this setting. SPECIFIC AIM 2: To determine the role of the GDNF-RET system, if any, in determining the post-implantation fate of transplanted CNS-NSC. SPECIFIC AIM 3: To determine the physiological/functional effects of transplanted CNS-NSC on the gastrointestinal motility of mice with targeted disruption of the neuronal nitric oxide synthase gene (nNOS -/-). The proposed studies will lay the foundation for the use of CNS-NSC and other sources of neural stem cells (i.e. embryonic stem cells, neural crest stem cells or human neural stem ceils) for cellular replacement therapy in the gastrointestinal tract.